Table of contents

Materials and Methods

Animal Collection and Experimental Procedure
Mussels, M. galloprovincialis, 3.5 to 4.5 cm shell length
and of undetermined sex were collected at low tide from
ten different sampling sites in the NW and NE of the Iberian
Peninsula in July 2005 for the field studies, and in a NE
location in September 2005 for the laboratory experiments.
Sampling sites in the NW were Sao Bartolomeu do Mar
(41°34’36’’N, 8°48’2’’W) (from now on referred as Sao Bartolomeu),
Aguiño (42°31’13’’N, 9°0’36’’W), Caldebarcos
(42°50’48’’N, 9°7’52’’W), Camelle (43°11’38’’N,
9°5’48’’W), and Segaño (43°27’21’’N, 8°18’34’’W). Sampling
sites in the NE were Muskiz (43°21’32’’N, 3°6’40’’W),
Arrigunaga (43°21’17’’N, 3°1’11’’W), Gorliz (43°25’7’’N,
2°56’51’’W), Mundaka (43°24’16’’N, 2°41’43’’W), and
Hondarribia (43°22’40’’N, 1°47’24’’W). Mussels for the
laboratory experiment were collected from Mundaka
(43°24’16’’N, 2°41’43’’W), a relatively clean location in the
mouth of the Biosphere Reserve of Urdaibai estuary (Orbea and Cajaraville, 2006). Sampling sites are summarized in
Figure 1.

Mussels collected from Mundaka for the laboratory experiment
were acclimatized in the laboratory for 15 days
and afterwards divided in three high-density polyethylene
tanks at a mussel density of one mussel per three liters of
seawater. Water temperature was kept at 20 °C, salinity at 35‰ and oxygen levels above 6 mg/L by constant aeration.
A photoperiod of 11 hours was set and commercial food
(JBL KorallFluid, JBL BmgH & Co. KG, Neuhofen, Germany)
provided every day. The heavy fuel oil that is similar
to that spilled by the Prestige (IFO 380, marine fuel RMG
35-ISO 8217) was provided by the Vigo Technical Office
Against Accidental Marine Spills (University of Vigo, Spain).
Oily sediments were prepared by mixing 150 mL oil with 5
kg gravel, and 6 kg sand, and placed on the bottom of the
tanks. Weathered fuel oil (WF) was obtained by letting the
sediment stand in a water-filled tank during two and a half
months. Fresh fuel oil (FF) was obtained by adding the sediment
to a water filled tank precisely before the exposure
started. Exposure to FF tried to mimic the situation in the
most affected areas in the NW immediately after the
Prestige’s oil spill, whereas exposure to WF would mimic
the situation in any of the sampling sites months after the
spill. Mussels were also kept in a control tank where no oil
was added.

For our experiments, four mussels were collected from
each sampling site and eight from each tank: four after two
days of exposure, and four after 16 days had passed. Digestive
glands were immediately dissected out and frozen in
liquid nitrogen in situ in all the cases, and kept at - 80 °C
until the proteomics analysis.

Proteins from the eluted fractions were then precipitated
by the addition of 20% trichloroacetic acid in 100% cold
acetone containing 0.07% β-mercaptoethanol, and the precipitate
was washed with 100% cold acetone containing
0.07% β-mercaptoethanol. Precipitated proteins were solubilized
in a solubilization buffer described by Rabilloud with
some modifications (Rabilloud, 1998; Amelina et al., 2007),
alkylated with 30 mM iodoacetamide (IAA) in darkness and
mixed with a rehydration buffer previous to the
isoelectrofocusing (IEF) step. Proteins (300 µg) were loaded
in the PROTEAN® IEF Cell (BioRad Laboratories) tray and IPG strips (11 cm, pH range of 4-7, BioRad Laboratories)
placed on top. The following program was followed:
passive rehydration for 12 h at 50 V and 20 °C, 250 V for
15 min, rapid voltage ramping to obtain 8,000 V and a final
focusing at 8,000 V until 35,000 V.h were achieved. The
focusing was held at 500 V until strips were removed from
the tray. In all the steps, a maximum current limit of 50 µA
per strip was established. IPG strips were first reduced (1%
dithiothreitol (w/v)), and then alkylated (4% IAA (w/v)) in
an equilibration buffer (Amelina et al., 2007) previous to
SDS-PAGE.

Equilibrated IPG strips were laid on top of homogeneous
12.5% Tris-HCl Criterion™ Precast Gels (BioRad Laboratories)
and SDS-PAGE run at 120 V. 2- DE gels were fixed
and stained with CBB G-250 for 12-18 h. Distained 2-DE
gels were scanned in a UMAX Image Scanner (Amersham
Biosciences) and analyzed by ImageMaster™ 2D Platinum
6.0 (Amersham Biosciences). 2-DE gel images were
cropped, spots automatically detected, wrong detections
manually corrected and finally the volume % (vol%) of each
spot calculated based on the total spot volume in each gel.
A master gel was chosen for each sampling site and exposure
group. Spots from the rest of the three gels inside each
sampling site/group were then matched to the master gel.
Higher-level match-sets were constructed between master
gels. Image analyses of the field study and the laboratory
exposure were separately performed, but their highest-level
master images were finally matched between them.

Statistical Analysis

Vol% data was exported to SAS® 9.1.9 (SAS Institute
Inc., Cary, NC, USA) and MATLAB® 7.5.0 (The
MathWorks, Inc., Natick, MA, USA) for the statistical analyses.
In total, 468 spots were obtained in the match set from
the laboratory exposure experiment. Missing values in the
data set came from spots with intensities lower than the
detection limit of the image analyzer, or from spots absent
in the 2-DE gels, but not from an incorrect matching. Therefore,
zero values were input. In the few cases where the
missing value happened in a group with relatively high values,
the mean value of the three replicates from the group
was input.

Two-way ANOVA was performed on each spot separately
to extract those spots that differed among the groups,
based on the following linear effects model:

i = 1,2; j = 1, 2, 3; and k = 1, 2, 3, 4, where α is the time
effect average over treatments, β is the treatment effect
average over time, γ is the interaction effect, and ε is the variation within each group of 4 replicates, εijk ~ N (0, σij).
The response variable y is the value of the specific spot. On
account of performing multiple tests, there will necessarily
be a number of false positives. By use of the False Discovery
Rate (FDR) procedure (Hochberg and Benjamini, 1995)
we can protect against too many false positives. FDR was
set to 5 %.

PCA is a multivariate statistics technique that takes into
account a group of variables instead of focusing in one variable
at a time, as is the case for univariate analyses. PCA
was used to find out if there was any structure in the data
selected after the ANOVA and FRD analyses that could
explain differences among the exposure groups. A covariance
matrix where each spot was set as a variable and
each gel as an observation was used to extract the principal
components. In order to improve the PCA outcome, several
spots were removed from the dataset.

Finally, the vol% of the selected spots was obtained from
the field experiment data. A putative group membership for
the different sampling sites was obtained based on the new
variable’s proximity to the experimental variables that were
separated by the PCA.